Die Bedeutung von microRNAs für die Funktion von Endothelzellen

Dicer and Drosha are the major enzymes involved in microRNA processing. Using siRNA targeting Dicer and Drosha, thereby downregulating a substantial number of microRNAs in EC, we demonstrate a crucial role of both enzymes in angiogenic processes. Interestingly, Dicer inhibition exerts more profound effects on processes like migration and viability of EC in comparison to Drosha inhibition. Moreover, Dicer effects in vivo angiogenesis, a process which is unaffected by Drosha. This discrepancy might be partially due to the involvement of Dicer in other cellular processes like heterochromatin formation and to the fact that Dicer and Drosha target mainly different subsets of microRNAs. In addition, we identified miR-92a as a novel endogenous repressor of the angiogenic program in EC, which impairs their angiogenic functions in vitro and in vivo. Consistent with these data, blocking miR-92a by systemic infusion of antagomirs enhances neovascularization and functional recovery after ischemia in vivo. At first sight, the anti-angiogenic function of miR-92a in EC appears to contradict the previously identified anti-apoptotic and pro-angiogenic activities of the miR-17~92 cluster in tumor cells. However, this apparent discrepancy might be well rationalized by a predominant function of miR-18a and miR-19a in tumor cells, which are responsible for the tumorigenic and non-cell autonomous pro-angiogenic functions of the miR-17~92 cluster. Instead, miR-92a expression is specifically upregulated in ischemic tissues and appears to cell-autonomously repress the angiogenic potential of EC. Among the various targets and verified regulated genes identified by microarray, we confirmed the downregulation of Integrin a5 in vitro and in vivo. The relevance of this miR-92a target is evidenced by severe vascular defects in the absence of Integrin a5. In addition, endothelial miR-92a interferes with the expression pattern of genes controlling key EC functions at various levels, some of which, e.g. eNOS, might be secondarily affected by directly targeted genes. Obviously, our data do not formally exclude effects of antagomir-92a on perivascular and other cell types, but surely include effects on EC. Regardless of this, the capacity of miR-92a to target various downstream effectors might be an advantage of miRNA-based therapeutic strategies and may overcome the limited therapeutic capacity of single growth factor or single gene therapies in ischemic diseases, since the highly organized process of vessel growth, maturation and functional maintenance is well known to require the fine-tuned regulation of a set of genes.Angiogenese, die Bildung von Kapillaren aus bereits existierenden Gefäßen, ist ein wichtiger physiologischer Prozess zur Wundheilung und Wiederherstellung des Blutflusses nach Verletzung. Das Ziel der vorliegenden Arbeit war die genaue Untersuchung der Rolle von microRNAs in angiogenen Prozessen. MicroRNAs sind kleine, einzelsträngige RNA Moleküle, welche die Genexpression durch Bindung an die messenger RNA (mRNA) von Zielgenen und darauffolgenden Degradierung der mRNA oder Repression der Translation regulieren. Die Inhibition der microRNA-prozessierenden Enzyme Dicer und Drosha in Endothelzellen führt zu einer Dysregulation der microRNA Expression und zu einer signifikanten Reduktion der Angiogenese in vitro. Im Gegensatz zu Drosha, dessen Inhibition keinen Effekt auf die in vivo Angiogenese zeigt, führt die Inhibition von Dicer auch in einem in vivo Angiogenese-Modell zu einer deutliche reduzierten Einsprossung von Gefäßen. Zusammenfassend sprechen diese Daten für eine wichtige Rolle von microRNAs in der Endothelzellbiologie. Der zweite Teil der vorliegenden Arbeit beschränkte sich auf die Untersuchung einer, in Endothelzellen hoch exprimierten microRNA, miR-92a. MiR-92a ist ein Mitglied des miR-17-92 Clusters, für den bereits eine Rolle in der Tumorangiogenese beschrieben ist. Die Überexpression der miR-92a in Endothelzellen führt zu einer signifikanten Hemmung der Angiogenese, sowie reduzierter Adhäsion und Migration auf Fibronektin. Zudem wird die Einsprossung von Gefäßen in Matrigel Plugs deutlich gehemmt. In Übereinstimmung hierzu führt die systemische Hemmung der miR-92a mit Hilfe von modifizierten antisense Oligoribonukleotiden (Antagomir-92a) im Mausmodell zu einer Stimulation der Einsprossung von Gefäßen in Matrigel Plugs. In klinisch relevanten Modellen, wie dem Hinterlaufischämie-Modell und dem akuten Myokardinfarkt-Modell, führt die Behandlung mit Antagomir-92a zu einer funktionelle Verbesserung. Immunohistologische Analysen ergaben, dass in den Antagomir-92a behandelten Tieren sowohl die Anzahl der Kapillaren als auch der größeren Gefäße deutlich erhöht ist. Obwohl wir einen direkten Effekt der Antagomir-92a Behandlung auf die Myozyten zum jetzigen Zeitpunkt nicht auschließen können, weisen unsere Daten definitiv auf eine Stimulation der Angiogenese hin. Nachdem wir sowohl in vitro als auch in vivo zeigen konnten, dass miR-92a maßgeblich an angiogenen Prozessen beteiligt ist, stellten wir uns die Frage nach den zugrunde liegenden Zielgenen. Es zeigte sich, dass miR-92a neben einer größeren Anzahl pro-angiogener Faktoren die Integrin Untereinheit a5 sowohl in vitro als auch in vivo reguliert. Desweiteren konnten wir mit Hilfe eines Luciferase Assays die direkte Regulation von Integrin a5 durch miR-92a zeigen. Zusammenfassend deuten diese Daten daraufhin, dass Integrin a5 ein Schlüsselregulator der Antagomir-92a-vermittelten Angiogenese ist und somit unmittelbar an der Verbesserung nach Hinterlaufischämie und akutem Myokardinfarkt beteiligt ist

Vascular microRNAs

MicroRNAs are endogenously expressed small non-coding RNAs that regulate gene expression on the posttranscriptional level. During the last years microRNAs have emerged as key regulators of several physiological and pathophysiological processes in the vascular wall. Endothelial cell functions and angiogenesis are critically regulated by microRNAs such as miR-126 and the miR-17-92 cluster in vitro and in vivo. Tumor angiogenesis is additionally controlled by miR-296 and miR-378. MicroRNAs also regulate smooth muscle cell phenotypes and control neointima formation and atherosclerosis. In this respect, miR-143 and miR-145 have been shown to play a crucial role. In this review, we summarize the role of microRNAs and their target genes in endothelial and smooth muscle cells and discuss their applicability as drug targets

<資料>土耳古和議法と瑞西和議法

MicroRNAs (miRNAs, miRs) emerged as key regulators of gene expression. Germline hemizygous deletion of the gene that encodes the miR-17∼92 miRNA cluster was associated with microcephaly, short stature and digital abnormalities in humans. Mice deficient for the miR-17∼92 cluster phenocopy several features such as growth and skeletal development defects and exhibit impaired B cell development. However, the individual contribution of miR-17∼92 cluster members to this phenotype is unknown. Here we show that germline deletion of miR-92a in mice is not affecting heart development and does not reduce circulating or bone marrow-derived hematopoietic cells, but induces skeletal defects. MiR-92a-/- mice are born at a reduced Mendelian ratio, but surviving mice are viable and fertile. However, body weight of miR-92a-/- mice was reduced during embryonic and postnatal development and adulthood. A significantly reduced body and skull length was observed in miR-92a-/- mice compared to wild type littermates. µCT analysis revealed that the length of the 5th mesophalanx to 5th metacarpal bone of the forelimbs was significantly reduced, but bones of the hindlimbs were not altered. Bone density was not affected. These findings demonstrate that deletion of miR-92a is sufficient to induce a developmental skeletal defect

MiR-92a^−/− mice survive at a reduced Mendelian ratio.

<p>(<b>A</b>) Representative agarose gel picture of PCR products (WT allele: 1718 bp; miR-92a knockout (KO) allele: 422 bp) for genotyping of WT, miR-92a^+/− and miR-92a^−/− mice. (<b>B</b>) MiR-92a expression in the heart of WT, miR-92a^+/− and miR-92a^−/− mice. (<b>C</b>) Expression of the miR-17∼92 cluster members miR-17, miR-18a, miR-19a, miR-20a and miR-19b in the heart of WT, miR-92a^+/− and miR-92a^−/− mice. (<b>D</b>) Observed as well as by Mendelian ratios predicted percentage of weaned WT, miR-92a^+/− and miR-92a^−/− mice derived from mating of miR-92a^+/− mice. Data are represented as mean ± SEM, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by one-way ANOVA; ^#<i>P</i><0.05 by Clopper-Pearson interval, ^$<i>P</i><0.01 by chi-square test.</p

Inhibition of miR-92a improves re-endothelialization and prevents neointima formation following vascular injury

AIMS: MicroRNA (miR)-92a is an important regulator of endothelial proliferation and angiogenesis after ischaemia, but the effects of miR-92a on re-endothelialization and neointimal lesion formation after vascular injury remain elusive. We tested the effects of lowering miR-92a levels using specific locked nucleic acid (LNA)-based antimiRs as well as endothelial-specific knock out of miR-92a on re-endothelialization and neointimal formation after wire-induced injury of the femoral artery in mice. METHODS AND RESULTS: MiR-92a was significantly up-regulated in neointimal lesions following wire-induced injury. Pre-miR-92a overexpression resulted in repression of the direct miR-92a target genes integrin α5 and sirtuin1, and reduced eNOS expression in vitro. MiR-92a impaired proliferation and migration of endothelial cells but not smooth muscle cells. In vivo, systemic inhibition of miR-92a expression with LNA-modified antisense molecules resulted in a significant acceleration of re-endothelialization of the denuded vessel area. Genetic deletion of miR-92a in Tie2-expressing cells, representing mainly endothelial cells, enhanced re-endothelialization, whereas no phenotype was observed in mice lacking miR-92a expression in haematopoietic cells. The enhanced endothelial recovery was associated with reduced accumulation of leucocytes and inhibition of neointimal formation 21 days after injury and led to the de-repression of the miR-92a targets integrin α5 and sirtuin1. CONCLUSION: Our data indicate that inhibition of endothelial miR-92a attenuates neointimal lesion formation by accelerating re-endothelialization and thus represents a putative novel mechanism to enhance the functional recovery following vascular injury

MicroRNA-27a/b controls endothelial cell repulsion and angiogenesis by targeting semaphorin 6A

MicroRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. miR-27 is expressed in endothelial cells, but the specific functions of miR-27b and its family member miR-27a are largely unknown. Here we demonstrate that overexpression of miR-27a and miR-27b significantly increased endothelial cell sprouting. Inhibition of both miR-27a and miR-27b impaired endothelial cell sprout formation and induced endothelial cell repulsion in vitro. In vivo, inhibition of miR-27a/b decreased the number of perfused vessels in Matrigel plugs and impaired embryonic vessel formation in zebrafish. Mechanistically, miR-27 regulated the expression of the angiogenesis inhibitor semaphorin 6A (SEMA6A) in vitro and in vivo and targeted the 3′-untranslated region of SEMA6A. Silencing of SEMA6A partially reversed the inhibition of endothelial cell sprouting and abrogated the repulsion of endothelial cells mediated by miR-27a/b inhibition, indicating that SEMA6A is a functionally relevant miR-27 downstream target regulating endothelial cell repulsion. In summary, we show that miR-27a/b promotes angiogenesis by targeting the angiogenesis inhibitor SEMA6A, which controls repulsion of neighboring endothelial cells

MicroRNA-34a regulates cardiac ageing and function

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition